Characterization of lysine acetylation in the peripheral subunit-binding domain of the E2 subunit of the pyruvate dehydrogenase-2-oxoglutarate dehydrogenase hybrid complex from Corynebacterium glutamicum.

In Corynebacterium glutamicum, pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (ODH) form a unique hybrid complex in which CgE1p and CgE1o are associated with the CgE2-CgE3 subcomplex. We analyzed the role of a lysine acetylation site in the peripheral subunit-binding domain of CgE2 in PDH and ODH functions. Acetylation-mimic substitution at Lys391 of CgE2 severely reduced the interaction of CgE2 with CgE1p and CgE3, but not with CgE1o, indicating the critical role of this residue in the assembly of CgE1p and CgE3 into the complex. It also suggested that Lys391 acetylation inhibited the binding of CgE1p and CgE3 to CgE2, thereby affecting PDH and ODH activities. Interestingly, the CgE2-K391R variant strain showed increased l-glutamate production and reduced pyruvate accumulation. Kinetic analysis suggested that the increased affinity of the K391R variant toward pyruvate might be advantageous for l-glutamate production.

In vitro reconstitution and characterization of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase hybrid complex from Corynebacterium glutamicum.

Pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (ODH) are critical enzymes in central carbon metabolism. In Corynebacterium glutamicum, an unusual hybrid complex consisting of CgE1p (thiamine diphosphate-dependent pyruvate dehydrogenase, AceE), CgE2 (dihydrolipoamide acetyltransferase, AceF), CgE3 (dihydrolipoamide dehydrogenase, Lpd), and CgE1o (thiamine diphosphate-dependent 2-oxoglutarate dehydrogenase, OdhA) has been suggested. Here, we elucidated that the PDH-ODH hybrid complex in C. glutamicum probably consists of six copies of CgE2 in its core, which is rather compact compared with PDH and ODH in other microorganisms that have twenty-four copies of E2. We found that CgE2 formed a stable complex with CgE3 (CgE2-E3 subcomplex) in vitro, hypothetically comprised of two CgE2 trimers and four CgE3 dimers. We also found that CgE1o exists mainly as a hexamer in solution and is ready to form an active ODH complex when mixed with the CgE2-E3 subcomplex. Our in vitro reconstituted system showed CgE1p- and CgE1o-dependent inhibition of ODH and PDH, respectively, actively supporting the formation of the hybrid complex, in which both CgE1p and CgE1o associate with a single CgE2-E3. In gel filtration chromatography, all the subunits of CgODH were eluted in the same fraction, whereas CgE1p was eluted separately from CgE2-E3, suggesting a weak association of CgE1p with CgE2 compared with that of CgE1o. This study revealed the unique molecular architecture of the hybrid complex from C. glutamicum and the compact-sized complex would provide an advantage to determine the whole structure of the unusual hybrid complex.

Effect of lysine succinylation on the regulation of 2-oxoglutarate dehydrogenase inhibitor, OdhI, involved in glutamate production in Corynebacterium glutamicum.

In Corynebacterium glutamicum, the activity of the 2-oxoglutarate dehydrogenase (ODH) complex is negatively regulated by the unphosphorylated form of OdhI protein, which is critical for L-glutamate overproduction. We examined the potential impact of protein acylation at lysine (K)-132 of OdhI in C. glutamicum ATCC13032. The K132E succinylation-mimic mutation reduced the ability of OdhI to bind OdhA, the catalytic subunit of the ODH complex, which reduced the inhibition of ODH activity. In vitro succinylation of OdhI protein also reduced the ability to inhibit ODH, and the K132R mutation blocked the effect. These results suggest that succinylation at K132 may attenuate the OdhI function. Consistent with these results, the C. glutamicum mutant strain with OdhI-K132E showed decreased L-glutamate production. Our results indicated that not only phosphorylation but also succinylation of OdhI protein may regulate L-glutamate production in C. glutamicum.